The androgen-induced development of the male phenotype and reproductive function is mediated by the androgen receptor (AR), a nuclear transactivating factor that regulates the expression of specific genes. Mutations of the AR gene in 46,XY males cause the androgen insensitivity syndrome, the clinical manifestations of which include either the female phenotype, ambiguous genitalia, or phenotypically normal male with infertility. I propose to identify and analyze natural AR gene mutations in individuals with androgen insensitivity in order to characterize structure/function relationships of the AR and to better understand the mechanisms of this syndrome. I am particularly interested in evaluating mutations in the N-terminal domain of the AR since this region is the least well characterized and appears to be essential for transcriptional activation. Mutations will be identified by first amplifying the AR gene exons using the polymerase chain reaction. Large deletions are detected by gel electrophoresis while small mutations and single base mutations are screened by the GC clamp method. The DNA sequence of the mutated exons is determined by the dideoxy-mediated chain termination method. Natural mutations will be recreated by site directed mutagenesis of normal human AR complementary DNA, and the functional characteristics of the expressed receptors will be analyzed for steroid binding, DNA binding, and transcriptional activation. Evaluating the in vivo effects of the mutations on androgen binding and RNA turnover will be facilitated by culturing genital skin fibroblasts from these individuals.